bims-mascan 2019-10-27 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019–10–27
23 papers selected by
Giovanny Rodríguez Blanco, The Beatson Institute for Cancer Research

The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.
Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.
Characterization of Lipid Profiles after Dietary Intake of Polyunsaturated Fatty Acids Using Integrated Untargeted and Targeted Lipidomics.
Troubleshooting in Large-Scale LC-ToF-MS Metabolomics Analysis: Solving Complex Issues in Big Cohorts.
Proteomics and Metabolomics in Kidney Disease, including Insights into Etiology, Treatment, and Prevention.
Lipidomics: Current state of the art in a fast moving field.
Metabolomic profiling identifies pathways associated with minimal residual disease in childhood acute lymphoblastic leukaemia.
Plasma Metabolomics Identifies Lipid and Amino Acid Markers of Weight Loss in Patients with Upper Gastrointestinal Cancer.
Aberrant lipid metabolism as an emerging therapeutic strategy to target cancer stem cells: Concise review.
Inborn Errors of Metabolism in the Era of Untargeted Metabolomics and Lipidomics.
Mass spectrometric analysis of PTM dynamics using stable isotope labeled metabolic precursors in cell culture.
UPLC‑MS/MS‑based metabolomic characterization and comparison of pancreatic adenocarcinoma tissues using formalin‑fixed, paraffin‑embedded and optimal cutting temperature‑embedded materials.
Absolute quantification of RNA or DNA using acid hydrolysis and mass spectrometry.
Oncometabolites in renal cancer.
Glutamine Metabolism in Brain Tumors.
Epoxyeicosatrienoic acid (EET)-stimulated angiogenesis is mediated by epoxy hydroxyeicosatrienoic acids (EHETs) formed from COX-2.
The cytosolic form of aspartate aminotransferase is required for full activation of TOR complex 1 in fission yeast.
International Ring Trial of a High Resolution Targeted Metabolomics and Lipidomics Platform for Serum and Plasma Analysis.
A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues.
Mass Spectrometry-Based Analysis of Time-Resolved Proteome Quantification.
Applying mass spectrometry-based assays to explore gut microbial metabolism and associations with disease.
Using Cerebral Metabolites to Guide Precision Medicine for Subarachnoid Hemorrhage: Lactate and Pyruvate.
Supercritical fluid chromatography coupled to mass spectrometry for lipidomics.

Nature. 2019 Oct 21.

The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.

Kirill Bersuker, Joseph Hendricks, Zhipeng Li, Leslie Magtanong, Breanna Ford, Peter H Tang, Melissa A Roberts, Bingqi Tong, Thomas J Maimone, Roberto Zoncu, Michael C Bassik, Daniel K Nomura, Scott J Dixon, James A Olzmann.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, suggesting that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ), generating a lipophilic radical-trapping antioxidant (RTA) that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumor xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a new ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutics.

DOI: https://doi.org/10.1038/s41586-019-1705-2
Metabolites. 2019 Oct 18. pii: E235. [Epub ahead of print]9(10):

Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

Sidney Behringer, Victoria Wingert, Victor Oria, Anke Schumann, Sarah Grünert, Artur Cieslar-Pobuda, Stefan Kölker, Ann-Kathrin Lederer, Donald W Jacobsen, Judith Staerk, Oliver Schilling, Ute Spiekerkoetter, Luciana Hannibal.

  The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.

Keywords:  biomarker; glutathione; homocysteine; mass spectrometry; methionine metabolism; targeted metabolic profiling; thiol

DOI:  https://doi.org/10.3390/metabo9100235
Metabolites. 2019 Oct 21. pii: E241. [Epub ahead of print]9(10):

Characterization of Lipid Profiles after Dietary Intake of Polyunsaturated Fatty Acids Using Integrated Untargeted and Targeted Lipidomics.

Satoko Naoe, Hiroshi Tsugawa, Mikiko Takahashi, Kazutaka Ikeda, Makoto Arita.

  Illuminating the comprehensive lipid profiles after dietary supplementation of polyunsaturated fatty acids (PUFAs) is crucial to revealing the tissue distribution of PUFAs in living organisms, as well as to providing novel insights into lipid metabolism. Here, we performed lipidomic analyses on mouse plasma and nine tissues, including the liver, kidney, brain, white adipose, heart, lung, small intestine, skeletal muscle, and spleen, with the dietary intake conditions of arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) as the ethyl ester form. We incorporated targeted and untargeted approaches for profiling oxylipins and complex lipids such as glycerol (phospho) lipids, sphingolipids, and sterols, respectively, which led to the characterization of 1026 lipid molecules from the mouse tissues. The lipidomic analysis indicated that the intake of PUFAs strongly impacted the lipid profiles of metabolic organs such as the liver and kidney, while causing less impact on the brain. Moreover, we revealed a unique lipid modulation in most tissues, where phospholipids containing linoleic acid were significantly decreased in mice on the ARA-supplemented diet, and bis(monoacylglycero)phosphate (BMP) selectively incorporated DHA over ARA and EPA. We comprehensively studied the lipid profiles after dietary intake of PUFAs, which gives insight into lipid metabolism and nutrition research on PUFA supplementation.

Keywords:  arachidonic acid; dietary fat; fatty acid metabolism; lipidomics; mass spectrometry; omega-3 fatty acids

DOI:  https://doi.org/10.3390/metabo9100241
Metabolites. 2019 Oct 24. pii: E247. [Epub ahead of print]9(11):

Troubleshooting in Large-Scale LC-ToF-MS Metabolomics Analysis: Solving Complex Issues in Big Cohorts.

Juan Rodríguez-Coira, María I Delgado-Dolset, David Obeso, Mariana Dolores-Hernández, Guillermo Quintás, Santiago Angulo, Domingo Barber, Teresa Carrillo, María M Escribese, Alma Villaseñor.

  Metabolomics, understood as the science that manages the study of compounds from the metabolism, is an essential tool for deciphering metabolic changes in disease. The experiments rely on the use of high-throughput analytical techniques such as liquid chromatography coupled to mass spectrometry (LC-ToF MS). This hyphenation has brought positive aspects such as higher sensitivity, specificity and the extension of the metabolome coverage in a single run. The analysis of a high number of samples in a single batch is currently not always feasible due to technical and practical issues (i.e., a drop of the MS signal) which result in the MS stopping during the experiment obtaining more than a single sample batch. In this situation, careful data treatment is required to enable an accurate joint analysis of multi-batch data sets. This paper summarizes the analytical strategies in large-scale metabolomic experiments; special attention has been given to QC preparation troubleshooting and data treatment. Moreover, labeled internal standards analysis and their aim in data treatment, and data normalization procedures (intra- and inter-batch) are described. These concepts are exemplified using a cohort of 165 patients from a study in asthma.

Keywords:  LC-QToF-MS; asthma; large-scale; metabolomics; normalization

DOI:  https://doi.org/10.3390/metabo9110247
Clin J Am Soc Nephrol. 2019 Oct 21. pii: CJN.07420619. [Epub ahead of print]

Proteomics and Metabolomics in Kidney Disease, including Insights into Etiology, Treatment, and Prevention.

Ruth F Dubin, Eugene P Rhee.

  In this review of the application of proteomics and metabolomics to kidney disease research, we review key concepts, highlight illustrative examples, and outline future directions. The proteome and metabolome reflect the influence of environmental exposures in addition to genetic coding. Circulating levels of proteins and metabolites are dynamic and modifiable, and thus amenable to therapeutic targeting. Design and analytic considerations in proteomics and metabolomics studies should be tailored to the investigator's goals. For the identification of clinical biomarkers, adjustment for all potential confounding variables, particularly GFR, and strict significance thresholds are warranted. However, this approach has the potential to obscure biologic signals and can be overly conservative given the high degree of intercorrelation within the proteome and metabolome. Mass spectrometry, often coupled to up-front chromatographic separation techniques, is a major workhorse in both proteomics and metabolomics. High-throughput antibody- and aptamer-based proteomic platforms have emerged as additional, powerful approaches to assay the proteome. As the breadth of coverage for these methodologies continues to expand, machine learning tools and pathway analyses can help select the molecules of greatest interest and categorize them in distinct biologic themes. Studies to date have already made a substantial effect, for example elucidating target antigens in membranous nephropathy, identifying a signature of urinary peptides that adds prognostic information to urinary albumin in CKD, implicating circulating inflammatory proteins as potential mediators of diabetic nephropathy, demonstrating the key role of the microbiome in the uremic milieu, and highlighting kidney bioenergetics as a modifiable factor in AKI. Additional studies are required to replicate and expand on these findings in independent cohorts. Further, more work is needed to understand the longitudinal trajectory of select protein and metabolite markers, perform transomics analyses within merged datasets, and incorporate more kidney tissue-based investigation.

Keywords:  Metabolomics; albumins; biological products; biomarkers; chronic renal insufficiency; confounding factors (epidemiology); diabetic nephropathies; diabetic nephropathy; energy metabolism; environmental exposure; goals; kidney; machine learning; mass spectrometry; membranous glomerulonephritis; metabolome; microbiota; peptides; prognosis; proteome; proteomics; research personnel

DOI:  https://doi.org/10.2215/CJN.07420619
Wiley Interdiscip Rev Syst Biol Med. 2019 Oct 23. e1466

Lipidomics: Current state of the art in a fast moving field.

Valerie B O'Donnell, Kim Ekroos, Gerhard Liebisch, Michael Wakelam.

  Lipids are essential for all facets of life. They play three major roles: energy metabolism, structural, and signaling. They are dynamic molecules strongly influenced by endogenous and exogenous factors including genetics, diet, age, lifestyle, drugs, disease and inflammation. As precision medicine starts to become mainstream, there is a huge burgeoning interest in lipids and their potential to act as unique biomarkers or prognostic indicators. Lipids comprise a large component of all metabolites (around one-third), and our expanding knowledge about their dynamic behavior is fueling the hope that mapping their regulatory biochemical pathways on a systems level will revolutionize our ability to prevent, diagnose, and stratify major human diseases. Up to now, clinical lipid measurements have consisted primarily of total cholesterol or triglycerides, as a measure for cardiovascular risk and response to lipid lowering drugs. Nowadays, we are able to measure thousands of individual lipids that make up the lipidome. nuclear magnetic resonance spectrometry (NMR) metabolomics is also being increasingly used in large cohort studies where it can report on total levels of selected lipid classes, and relative levels of fatty acid saturation. To support the application of lipidomics research, LIPID MAPS was established in 2003, and since then has gone on to become the go-to resource for several lipid databases, lipid drawing tools, data deposition, and more recently lipidomics informatics tools, and a lipid biochemistry encyclopedia, LipidWeb. Alongside this, the recently established Lipidomics Standards Initiative plays a key role in standardization of lipidomics methodologies. This article is categorized under: Laboratory Methods and Technologies > Metabolomics Analytical and Computational Methods > Analytical Methods.

Keywords:  biomarkers; lipid; lipidomics; mass spectrometry; precision medicine

DOI:  https://doi.org/10.1002/wsbm.1466
EBioMedicine. 2019 Oct 17. pii: S2352-3964(19)30660-7. [Epub ahead of print]

Metabolomic profiling identifies pathways associated with minimal residual disease in childhood acute lymphoblastic leukaemia.

Jeremy M Schraw, Jacob J Junco, Austin L Brown, Michael E Scheurer, Karen R Rabin, Philip J Lupo.

   BACKGROUND: End-induction minimal residual disease (MRD) is the strongest predictor of relapse in paediatric acute lymphoblastic leukaemia (ALL), but an understanding of the biological pathways underlying early treatment response remains elusive. We hypothesized that metabolomic profiling of diagnostic bone marrow plasma could provide insights into the underlying biology of early treatment response and inform treatment strategies for high-risk patients.
METHODS: We performed global metabolomic profiling of samples from discovery (N = 93) and replication (N = 62) cohorts treated at Texas Children's Hospital. Next, we tested the cytotoxicity of drugs targeting central carbon metabolism in cell lines and patient-derived xenograft (PDX) cells.
FINDINGS: Metabolite set enrichment analysis identified altered central carbon and amino acid metabolism in MRD-positive patients from both cohorts at a 5% false discovery rate. Metabolites from these pathways were used as inputs for unsupervised hierarchical clustering. Two distinct clusters were identified, which were independently associated with MRD after adjustment for immunophenotype, cytogenetics, and NCI risk group. Three nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which reduce glycolytic/TCA cycle activities, demonstrated nanomolar-range cytotoxicity in B- and T-ALL cell lines and PDX cells.
INTERPRETATION: This study provides new insights into the role of central carbon metabolism in early treatment response and as a potential targetable pathway in high-risk disease.
FUNDING: American Society of Hematology; Baylor College of Medicine Department of Paediatrics; Cancer Prevention and Research Institute of Texas; the Lynch family; St. Baldrick's Foundation with support from the Micaela's Army Foundation; United States National Institutes of Health.

Keywords:  Acute lymphoblastic leukaemia; Epidemiology; Minimal residual disease; NAMPT; NAMPT inhibitors

DOI:  https://doi.org/10.1016/j.ebiom.2019.09.033
Cancers (Basel). 2019 Oct 19. pii: E1594. [Epub ahead of print]11(10):

Plasma Metabolomics Identifies Lipid and Amino Acid Markers of Weight Loss in Patients with Upper Gastrointestinal Cancer.

Janice Miller, Ahmed Alshehri, Michael I Ramage, Nathan A Stephens, Alexander B Mullen, Marie Boyd, James A Ross, Stephen J Wigmore, David G Watson, Richard J E Skipworth.

  Cachexia is a multifactorial wasting syndrome associated with high morbidity and mortality in patients with cancer. Diagnosis can be difficult and, in the clinical situation, usually relies upon reported weight loss. The 'omics' technologies allow us the opportunity to study the end points of many biological processes. Among these, blood-based metabolomics is a promising method to investigate the pathophysiology of human cancer cachexia and identify candidate biomarkers. In this study, we performed liquid chromatography mass spectrometry (LC/MS)-based metabolomics to investigate the metabolic profile of cancer-associated weight loss. Non-selected patients undergoing surgery with curative intent for upper gastrointestinal cancer were recruited. Fasting plasma samples were taken at induction of anaesthesia. LC/MS analysis showed that 6 metabolites were highly discriminative of weight loss. Specifically, a combination profile of LysoPC 18.2, L-Proline, Hexadecanoic acid, Octadecanoic acid, Phenylalanine and LysoPC 16:1 showed close correlation for eight weight-losing samples (≥5% weight loss) and nine weight-stable samples (<5%weight loss) between predicted and actual weight change (r = 0.976, p = 0.0014). Overall, 40 metabolites were associated with ≥5% weight loss. This study provides biological validation of the consensus definition of cancer cachexia (Fearon et al.) and provides feasible candidate markers for further investigation in early diagnosis and the assessment of therapeutic intervention.

Keywords:  cachexia; cancer; high resolution mass spectrometry; metabolomics

DOI:  https://doi.org/10.3390/cancers11101594
Stem Cells. 2019 Oct 24.

Aberrant lipid metabolism as an emerging therapeutic strategy to target cancer stem cells: Concise review.

Malini Visweswaran, Frank Arfuso, Sudha Warrier, Arun Dharmarajan.

  Emerging evidences in cancer metabolomics have identified reprogrammed metabolic pathways to be a major hallmark of cancer, amongst which deregulated lipid metabolism is a prominent field receiving increasing attention. Cancer stem cells (CSCs) comprise <0.1% of the tumour bulk and possess high self-renewal, tumour initiating properties, and are responsible for therapeutic resistance, disease recurrence, and tumour metastasis. Hence, it is imperative to understand the metabolic rewiring occurring in CSCs, especially their lipid metabolism, on which there have been recent reports. CSCs rely highly upon lipid metabolism for maintaining their stemness properties and fulfilling their biomass and energy demands, ultimately leading to cancer growth and invasion. Hence, in this review we will shed light on the aberrant lipid metabolism that CSCs exploit to boost their survival, which comprises upregulation in de novo lipogenesis, lipid droplet synthesis, lipid desaturation, and β-oxidation. Further, the metabolic regulators involved in the process, such as key lipogenic enzymes, are also highlighted. Finally, we also summarise the therapeutic strategies targeting the key regulators involved in CSCs' lipid metabolism, which thereby demonstrate the potential to develop powerful and novel therapeutics against the CSC lipid metabolome. © AlphaMed Press 2019 SIGNIFICANCE STATEMENT: This review describes the significance of altered lipid metabolism present in cancer stem cells (CSCs) originating from various cancers. It discusses the critical metabolic modifications occurring in CSCs that enable advanced growth and tumorigenesis through enhanced dependence on fatty acid synthesis and β-oxidation to fulfil their heightened energy and biomass requirements. Further, this review also summarises the various anti-cancer therapeutic strategies targeting CSC lipid metabolism.

Keywords:  Cancer stem cells; fatty acid synthesis; lipid metabolism; metabolic rewiring; β-oxidation

DOI:  https://doi.org/10.1002/stem.3101
Metabolites. 2019 Oct 21. pii: E242. [Epub ahead of print]9(10):

Inborn Errors of Metabolism in the Era of Untargeted Metabolomics and Lipidomics.

Israa T Ismail, Megan R Showalter, Oliver Fiehn.

  Inborn errors of metabolism (IEMs) are a group of inherited diseases with variable incidences. IEMs are caused by disrupting enzyme activities in specific metabolic pathways by genetic mutations, either directly or indirectly by cofactor deficiencies, causing altered levels of compounds associated with these pathways. While IEMs may present with multiple overlapping symptoms and metabolites, early and accurate diagnosis of IEMs is critical for the long-term health of affected subjects. The prevalence of IEMs differs between countries, likely because different IEM classifications and IEM screening methods are used. Currently, newborn screening programs exclusively use targeted metabolic assays that focus on limited panels of compounds for selected IEM diseases. Such targeted approaches face the problem of false negative and false positive diagnoses that could be overcome if metabolic screening adopted analyses of a broader range of analytes. Hence, we here review the prospects of using untargeted metabolomics for IEM screening. Untargeted metabolomics and lipidomics do not rely on predefined target lists and can detect as many metabolites as possible in a sample, allowing to screen for many metabolic pathways simultaneously. Examples are given for nontargeted analyses of IEMs, and prospects and limitations of different metabolomics methods are discussed. We conclude that dedicated studies are needed to compare accuracy and robustness of targeted and untargeted methods with respect to widening the scope of IEM diagnostics.

Keywords:  LC-MS; aminoacidemia.; lysosomal storage disease; mass spectrometry; mitochondrial disorders; organic aciduria; phenylketonuria

DOI:  https://doi.org/10.3390/metabo9100242
Analyst. 2019 Oct 25.

Mass spectrometric analysis of PTM dynamics using stable isotope labeled metabolic precursors in cell culture.

Alienke van Pijkeren, Rainer Bischoff, Marcel Kwiatkowski.

Biological organisms represent highly dynamic systems, which are continually exposed to environmental factors and always strive to restore steady-state homeostasis. Posttranslational modifications are key regulators with which biological systems respond to external stimuli. To understand how homeostasis is restored, it is important to study the kinetics of posttranslational modifications. In this review we discuss proteomic approaches using stable isotope labeled metabolic precursors to study dynamics of posttranslational modifications in cell culture.

DOI: https://doi.org/10.1039/c9an01258c
Int J Oncol. 2019 Oct 14.

UPLC‑MS/MS‑based metabolomic characterization and comparison of pancreatic adenocarcinoma tissues using formalin‑fixed, paraffin‑embedded and optimal cutting temperature‑embedded materials.

Di Feng, Jing Yuan, Qi Liu, Li Liu, Xu Zhang, Yali Wu, Yifan Qian, Liping Chen, Yan Shi, Mancang Gu.

The purpose of the present study was to compare metabolites from formalin‑fixed and paraffin‑embedded (FFPE) pancreatic tissue blocks with those identified in optimal cutting temperature (OCT)‑embedded pancreatic tissue blocks. Thus, ultra‑performance liquid chromatograph‑mass spectrometry/mass spectrometry‑based metabolic profiling was performed in paired frozen (n=13) and FFPE (n=13) human pancreatic adenocarcinoma tissue samples, in addition to their benign counterparts. A total of 206 metabolites were identified in both OCT‑embedded and FFPE tissue samples. The method feasibility was confirmed through reproducibility and a consistency assessment. Partial least‑squares discriminant analysis and heatmap analysis reliably distinguished tumor and normal tissue phenotypes. The expression of 10 compounds, including N‑acetylaspartate and creatinine, was significantly different in both OCT‑embedded and FFPE tumor samples. These ten compounds may be viable candidate biomarkers of malignant pancreatic tissues. The super‑categories to which they belonged exhibited no significant differences between FFPE and OCT‑embedded samples. Furthermore, purine, arginine and proline, and pyrimidine metabolism used a shared pathway found in both OCT‑embedded and FFPE tissue samples. These results supported the notion that metabolomic data acquired from FFPE pancreatic cancer specimens are reliable for use in retrospective and clinical studies.

DOI: https://doi.org/10.3892/ijo.2019.4898
Anal Chem. 2019 Oct 22.

Absolute quantification of RNA or DNA using acid hydrolysis and mass spectrometry.

Mark S Lowenthal, Eva Quittman, Karen W Phinney.

Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which can be then measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases, and by comparison to concentrations provided for commercial materials, or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop Spectrophotometry or established from droplet-digital PCR (ddPCR) techniques agree well to the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling SI-traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases, or mixtures.

DOI: https://doi.org/10.1021/acs.analchem.9b03625
Nat Rev Nephrol. 2019 Oct 21.

Oncometabolites in renal cancer.

Cissy Yong, Grant D Stewart, Christian Frezza.

The study of cancer metabolism has evolved vastly beyond the remit of tumour proliferation and survival with the identification of the role of 'oncometabolites' in tumorigenesis. Simply defined, oncometabolites are conventional metabolites that, when aberrantly accumulated, have pro-oncogenic functions. Their discovery has led researchers to revisit the Warburg hypothesis, first postulated in the 1950s, of aberrant metabolism as an aetiological determinant of cancer. As such, the identification of oncometabolites and their utilization in diagnostics and prognostics, as novel therapeutic targets and as biomarkers of disease, are areas of considerable interest in oncology. To date, fumarate, succinate, L-2-hydroxyglutarate (L-2-HG) and D-2-hydroxyglutarate (D-2-HG) have been characterized as bona fide oncometabolites. Extensive metabolic reprogramming occurs during tumour initiation and progression in renal cell carcinoma (RCC) and three oncometabolites - fumarate, succinate and L-2-HG - have been implicated in this disease process. All of these oncometabolites inhibit a superfamily of enzymes known as α-ketoglutarate-dependent dioxygenases, leading to epigenetic dysregulation and induction of pseudohypoxic phenotypes, and also have specific pro-oncogenic capabilities. Oncometabolites could potentially be exploited for the development of novel targeted therapies and as biomarkers of disease.

DOI: https://doi.org/10.1038/s41581-019-0210-z
Cancers (Basel). 2019 Oct 24. pii: E1628. [Epub ahead of print]11(11):

Glutamine Metabolism in Brain Tumors.

Siva Kumar Natarajan, Sriram Venneti.

  Altered metabolism is a hallmark of cancer cells. Tumor cells rewire their metabolism to support their uncontrolled proliferation by taking up nutrients from the microenvironment. The amino acid glutamine is a key nutrient that fuels biosynthetic processes including ATP generation, redox homeostasis, nucleotide, protein, and lipid synthesis. Glutamine as a precursor for the neurotransmitter glutamate, and plays a critical role in the normal functioning of the brain. Brain tumors that grow in this glutamine/glutamate rich microenvironment can make synaptic connections with glutamatergic neurons and reprogram glutamine metabolism to enable their growth. In this review, we examine the functions of glutamate/glutamine in the brain and how brain tumor cells reprogram glutamine metabolism. Altered glutamine metabolism can be leveraged to develop non-invasive imaging strategies and we review these imaging modalities. Finally, we examine if targeting glutamine metabolism could serve as a therapeutic strategy in brain tumors.

Keywords:  brain tumor; glutamate; glutamine; imaging; metabolism; redox homeostasis; therapy

DOI:  https://doi.org/10.3390/cancers11111628
J Lipid Res. 2019 Oct 22. pii: jlr.M094219. [Epub ahead of print]

Epoxyeicosatrienoic acid (EET)-stimulated angiogenesis is mediated by epoxy hydroxyeicosatrienoic acids (EHETs) formed from COX-2.

Amy A Rand, Anita Rajamani, Sean D Kodani, Todd R Harris, Lukas Schlatt, Bogdan Barnych, Anthony G Passerini, Bruce D Hammock.

  Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs elicit endothelial angiogenic activity linked to tumor growth in various cancer models that can be attenuated in vivo by COX-2 inhibitors. This study further defines the relationship between endothelial EET metabolism and COX-2 in promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential, in the presence and absence of a COX-2 inducer (PDBu). Although PDBu itself was potent in increasing angiogenic markers, 8,9-EET in combination with PDBu elicited a 1.3-fold larger response than 8,9-EET alone, and compared to PDBu. Contributing to this response were 8,9-EET metabolites formed from COX-2, the 11-hydroxy-8,9-EET (8,9,11-EHET) and 15-hydroxy-8,9-EET (8,9,15-EHET). When exogenously dosed into HAEC, synthetic 8,9,11-EHET enhanced angiogenesis at all concentrations tested, whereas 8,9,15-EHET was inactive. Tube formation by 8,9,11-EHET was independent of PI3K-Akt, p38 MAPK, and MEK signaling. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in endothelium through formation of 8,9,11-EHET. This lipid mediator may play a role in regulating physiological angiogenesis, and it may be especially important under circumstances where COX-2 is induced, such as in cancer tumor growth and inflammation. Finally, the generation of 8, 9, 11-EHET from 8, 9-EET may help explain why EETs are weakly angiogenic under some conditions yet block tumor growth under other conditions.

Keywords:  Arachidonic acid; Cancer; Cyclooxygenase; Endothelial cells; Mass spectrometry; angiogenesis; epoxyeicosatrienoic acid; metabolism; soluble epoxide hydrolase

DOI:  https://doi.org/10.1194/jlr.M094219
J Biol Chem. 2019 Oct 22. pii: jbc.RA119.010101. [Epub ahead of print]

The cytosolic form of aspartate aminotransferase is required for full activation of TOR complex 1 in fission yeast.

Sophie Reidman, Adiel Cohen, Martin Kupiec, Ronit Weisman.

  The evolutionarily conserved TOR complex 1 (TORC1) activates cell growth and proliferation in response to nutritional signals. In the fission yeast Schizosaccharomyces pombe, TORC1 is essential for vegetative growth, and its activity is regulated in response to nitrogen quantity and quality. Yet, how TORC1 senses nitrogen is poorly understood. Rapamycin, a specific TOR inhibitor, inhibits growth in S. pombe only under conditions in which the activity of TORC1 is compromised. In a genetic screen for rapamycin-sensitive mutations, we isolated caa1-1, a loss-of-function mutation of the cytosolic form of aspartate aminotransferase (Caa1). We demonstrate that loss of caa1 + partially mimics loss of TORC1 activity and that Caa1 is required for full TORC1 activity. Disruption of caa1 + resulted in aspartate auxotrophy, a finding that prompted us to assess the role of aspartate in TORC1 activation. We found that the amino acids glutamine, asparagine, arginine, aspartate, and serine activate TORC1 most efficiently following nitrogen starvation. The glutamine synthetase inhibitor L-methionine sulfoximine (MSX) abolished the ability of asparagine, arginine, aspartate, or serine, but not that of glutamine, to induce TORC1 activity, consistent with a central role for glutamine in activating TORC1. Neither addition of aspartate nor addition of glutamine restored TORC1 activity in caa1-deleted cells or in cells carrying a Caa1 variant with a catalytic-site substitution, suggesting that the catalytic activity of Caa1 is required for TORC1 activation. Taken together, our results reveal the contribution of the key metabolic enzyme Caa1 to TORC1 activity in S. pombe.

Keywords:  Caa1; Psk1; S6 kinase; Schizosaccharomyces pombe; TOR complex (TORC); aspartate amino acid transferase; glutamine synthase; nitrogen sensing; nutrient signaling; serine/threonine protein kinase; target of rapamycin (TOR)

DOI:  https://doi.org/10.1074/jbc.RA119.010101
Anal Chem. 2019 Oct 22.

International Ring Trial of a High Resolution Targeted Metabolomics and Lipidomics Platform for Serum and Plasma Analysis.

J Will Thompson, Kendra J Adams, Jerzy Adamski, Yasmin Asad, David Borts, John A Bowden, Gregory Byram, Viet D Dang, Warwick B Dunn, Facundo M Fernandez, Oliver Fiehn, David A Gaul, Andreas Huhmer, Anastasia Kalli, Therese Koal, Stormy Koeniger, Rupasri Mandal, Florian Meier, Fuad J Naser, Donna O'Neil, Akos Pal, Gary J Patti, Hai Pham-Tuan, Cornelia Prehn, Florence I Raynaud, Tong Shen, Andrew D Southam, Lisa St John-Williams, Karolina Sulek, Catherine G Vasilopoulou, Mark R Viant, Catherine L Winder, David S Wishart, Lun Zhang, Jiamin Zheng, M Arthur Moseley.

A challenge facing metabolomics in the analysis of large human cohorts is the cross-laboratory comparability of quantita-tive metabolomics measurements. In this study, 14 laboratories analyzed various blood specimens using a common ex-perimental protocol provided with Biocrates AbsoluteIDQ p400HR kit, to quantify up to 408 metabolites. The specimens included human plasma and serum from male and female donors, mouse and rat plasma as well as NIST SRM 1950 refer-ence plasma. The metabolite classes covered range from polar (e.g. amino acids and biogenic amines), to nonpolar (e.g. diacyl- and triacyl-glycerols), and span 11 common metabolite classes. The manuscript describes a strict system suita-bility testing (SST) criteria used to evaluate each laboratory's readiness to perform the assay, and provides the SST Sky-line documents for public dissemination. The study found approximately 250 metabolites were routinely quantified in the sample types tested, using Orbitrap instruments. Inter-laboratory variance for the NIST SRM-1950 has a median of 10% for amino acids, 24% for biogenic amines, 38% for acylcarnitines, 25% for glycerolipids, 23% for glycerophospho-lipids, 16% for cholesteryl esters, 15% for sphingolipids, and 9% for hexoses. Comparing to consensus values for NIST SRM-1950, nearly 80% of comparable analytes demonstrated bias of <50% from the reference value. The findings of this study result in recommendations of best practices for system suitability, quality control, and calibration. We demon-strate that with appropriate controls, high-resolution metabolomics can provide accurate results with good precision across laboratories, and the p400HR therefore is a reliable approach for generating consistent and comparable metabo-lomics data.

DOI: https://doi.org/10.1021/acs.analchem.9b02908
Biosci Rep. 2019 Oct 30. pii: BSR20192502. [Epub ahead of print]39(10):

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues.

Avishek Ghosh, Sanjeev Sharma, Dhananjay Shinde, Visvanathan Ramya, Padinjat Raghu.

  Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP (18O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

Keywords:  PIP4K; lipid kinase; mass spectrometry; phosphatidylinositol; phosphatidylinositol 5 phosphate

DOI:  https://doi.org/10.1042/BSR20192502
Proteomics. 2019 Oct 25. e1800425

Mass Spectrometry-Based Analysis of Time-Resolved Proteome Quantification.

Alberto Valdés, Sara Bergström Lind.

  The aspect of time is essential in biological processes and thus it is important to be able to monitor signaling molecules through time. Proteins are key players in cellular signaling and they respond to many stimuli and change their expression in many time-dependent processes. Mass spectrometry is an important tool for studying proteins, including their posttranslational modifications and their interaction partners - both in qualitative and quantitative ways. In order to distinguish the different trends over time, proteins, modification sites and interacting proteins must be compared between different time points, and therefore relative quantification is preferred. In this review, we discuss the progress and challenges for mass spectrometry-based analysis of time-resolved proteome dynamics. Further, aspects on model systems, technologies, sampling frequencies and presentation of the dynamic data are also discussed. This article is protected by copyright. All rights reserved.

Keywords:  comparative proteomics; interactomics; mass spectrometry -LC-MS/MS; post-translational modification analysis; protein dynamics; time-resolved proteomics

DOI:  https://doi.org/10.1002/pmic.201800425
Clin Chem Lab Med. 2019 Oct 22. pii: /j/cclm.ahead-of-print/cclm-2019-0974/cclm-2019-0974.xml. [Epub ahead of print]

Applying mass spectrometry-based assays to explore gut microbial metabolism and associations with disease.

Liam M Heaney.

  The workings of the gut microbiome have gained increasing interest in recent years through the mounting evidence that the microbiota plays an influential role in human health and disease. A principal focus of this research seeks to further understand the production of metabolic by-products produced by bacteria resident in the gut, and the subsequent interaction of these metabolites on host physiology and pathophysiology of disease. Gut bacterial metabolites of interest are predominately formed via metabolic breakdown of dietary compounds including choline and ʟ-carnitine (trimethylamine N-oxide), amino acids (phenol- and indole-containing uremic toxins) and non-digestible dietary fibers (short-chain fatty acids). Investigations have been accelerated through the application of mass spectrometry (MS)-based assays to quantitatively assess the concentration of these metabolites in laboratory- and animal-based experiments, as well as for direct circulating measurements in clinical research populations. This review seeks to explore the impact of these metabolites on disease, as well as to introduce the application of MS for those less accustomed to its use as a clinical tool, highlighting pertinent research related to its use for measurements of gut bacteria-mediated metabolites to further understand their associations with disease.

Keywords:  TMAO; biomarker; gut microbiome; mass spectrometry; short-chain fatty acids; uremic toxins

DOI:  https://doi.org/10.1515/cclm-2019-0974
Metabolites. 2019 Oct 23. pii: E245. [Epub ahead of print]9(11):

Using Cerebral Metabolites to Guide Precision Medicine for Subarachnoid Hemorrhage: Lactate and Pyruvate.

Kaneez Zahra, Neethu Gopal, William D Freeman, Marion T Turnbull.

  Subarachnoid hemorrhage (SAH) is one of the deadliest types of strokes with high rates of morbidity and permanent injury. Fluctuations in the levels of cerebral metabolites following SAH can be indicators of brain injury severity. Specifically, the changes in the levels of key metabolites involved in cellular metabolism, lactate and pyruvate, can be used as a biomarker for patient prognosis and tailor treatment to an individual's needs. Here, clinical research is reviewed on the usefulness of cerebral lactate and pyruvate measurements as a predictive tool for SAH outcomes and their potential to guide a precision medicine approach to treatment.

Keywords:  cerebral metabolism; delayed cerebral ischemia; lactate; lactate-pyruvate ratio; precision medicine; pyruvate; subarachnoid hemorrhage; vasospasm

DOI:  https://doi.org/10.3390/metabo9110245
J Mass Spectrom. 2019 Oct 25.

Supercritical fluid chromatography coupled to mass spectrometry for lipidomics.

Céline Chollet, Stéphanie Boutet-Mercey, Laboureur Laurent, Carlos Rincon, Marie Méjean, Juliette Jouhet, François Fenaille, Benoit Colsch, David Touboul.

Supercritical fluid chromatography (SFC) has experienced a particular revival in recent years thanks to the development of robust and efficient commercial systems. Due to its physico-chemical properties, supercritical carbon dioxide (CO2 ) mixed with co-solvents and additives is particularly suitable for SFC to allow the elution of compounds of different polarity, and more particularly complex lipids. Hyphenation with mass spectrometry (MS) is increasingly described in the literature but still requires many further developments in order to be as user-friendly as coupling with liquid chromatography. The basic concepts of SFC and MS hyphenation will be first considered. Then a representative example of method development in lipidomics will be introduced. In conclusion, the challenges and future needs in this field of research will be discussed.

DOI: https://doi.org/10.1002/jms.4445